scholarly journals PAR1b Promotes Cell–Cell Adhesion and Inhibits Dishevelled-mediated Transformation of Madin-Darby Canine Kidney Cells

2006 ◽  
Vol 17 (8) ◽  
pp. 3345-3355 ◽  
Author(s):  
Maya Elbert ◽  
David Cohen ◽  
Anne Müsch
Keyword(s):  
Cell Adhesion ◽  
Epithelial Cells ◽  
Wnt Signaling ◽  
Mdck Cells ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
E Cadherin ◽  
Cell Cell

Mammalian Par1 is a family of serine/threonine kinases comprised of four homologous isoforms that have been associated with tumor suppression and differentiation of epithelial and neuronal cells, yet little is known about their cellular functions. In polarizing kidney epithelial (Madin-Darby canine kidney [MDCK]) cells, the Par1 isoform Par1b/MARK2/EMK1 promotes the E-cadherin–dependent compaction, columnarization, and cytoskeletal organization characteristic of differentiated columnar epithelia. Here, we identify two functions of Par1b that likely contribute to its role as a tumor suppressor in epithelial cells. 1) The kinase promotes cell–cell adhesion and resistance of E-cadherin to extraction by nonionic detergents, a measure for the association of the E-cadherin cytoplasmic domain with the actin cytoskeleton, which is critical for E-cadherin function. 2) Par1b attenuates the effect of Dishevelled (Dvl) expression, an inducer of wnt signaling that causes transformation of epithelial cells. Although Dvl is a known Par1 substrate in vitro, we determined, after mapping the PAR1b-phosphorylation sites in Dvl, that PAR1b did not antagonize Dvl signaling by phosphorylating the wnt-signaling molecule. Instead, our data suggest that both proteins function antagonistically to regulate the assembly of functional E-cadherin–dependent adhesion complexes.

scholarly journals Par1b Promotes Hepatic-type Lumen Polarity in Madin Darby Canine Kidney Cells via Myosin II- and E-Cadherin–dependent Signaling

2007 ◽  
Vol 18 (6) ◽  
pp. 2203-2215 ◽  
Author(s):  
David Cohen ◽  
Yuan Tian ◽  
Anne Müsch
Keyword(s):  
Cell Adhesion ◽  
Mdck Cells ◽  
Myosin Ii ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Luminal Compartment ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
E Cadherin ◽  
Cell Cell

Kidney-derived Madin Darby canine kidney (MDCK) cells form lumina at their apices, and target luminal proteins to an intracellular vacuolar apical compartment (VAC) when prevented from polarizing. Hepatocytes, by contrast, organize their luminal surfaces (the bile canaliculi; BC) between their lateral membranes, and, when nonpolarized, they display an intracellular luminal compartment that is distinct from the VACs of MDCK cells. Overexpression of the serine/threonine kinase Par1b/EMK1/MARK2 induces BC-like lateral lumina and a hepatic-type intracellular luminal compartment in MDCK cells, suggesting a role for Par1b in the branching decision between kidney- and hepatic-type epithelial phenotypes. Here, we report that Par1b promotes lateral lumen polarity in MDCK cells independently of Ca2+-mediated cell–cell adhesion by inhibiting myosin II in a rho kinase-dependent manner. Polarization was inhibited by E-cadherin depletion but promoted by an adhesion-defective E-cadherin mutant. By contrast, apical surface formation in control MDCK cells required Ca2+-dependent cell–cell adhesion, but it occurred in the absence of E-cadherin. We propose that E-cadherin, when in an adhesion-incompetent state at the lateral domain, serves as targeting patch for the establishment of lateral luminal surfaces. E-cadherin depletion also reverted the hepatic-type intracellular luminal compartment in Par1b-MDCK cells to VACs characteristic of control MDCK cells, indicating a novel link between E-cadherin and luminal protein targeting.

scholarly journals Modulation of fodrin (membrane skeleton) stability by cell-cell contact in Madin-Darby canine kidney epithelial cells.

1987 ◽  
Vol 104 (6) ◽  
pp. 1527-1537 ◽  
Author(s):  
W J Nelson ◽  
P J Veshnock
Keyword(s):  
Membrane Proteins ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Membrane Skeleton ◽  
Cell Contact ◽  
Madin Darby Canine Kidney ◽  
The Stability ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
Cell Cell

During growth of Madin-Darby canine kidney (MDCK) epithelial cells, there is a dramatic change in the stability, biophysical properties, and distribution of the membrane skeleton (fodrin) which coincides temporally and spatially with the development of the polarized distribution of the Na+, K+-ATPase, a marker protein of the basolateral domain of the plasma membrane. These changes occur maximally upon the formation of a continuous monolayer of cells, indicating that extensive cell-cell contact may play an important role in the organization of polarized MDCK cells (Nelson, W. J., and P. J. Veshnock, 1986, J. Cell Biol., 103:1751-1766). To directly analyze the role of cell-cell contact in these events, we have used an assay in which the organization of fodrin and membrane proteins is analyzed in confluent monolayers of MDCK cells in the absence or presence of cell-cell contact by adjusting the concentration Ca++ in the growth medium. Our results on the stability and solubility properties of fodrin reported here show directly that there is a positive correlation between cell-cell contact and increased stability and insolubility of fodrin. Furthermore, we show that fodrin can be recruited from an unstable pool of protein to a stable pool during induction of cell-cell contact; significantly, the stabilization of fodrin is not affected by the addition of cyclohexamide, indicating that proteins normally synthesized during the induction of cell-cell contact are not required. Together these results indicate that cell-cell contact may play an important role in the development of polarity in MDCK cells by initiating the formation of a stable, insoluble matrix of fodrin with preexisting (membrane) proteins at the cell periphery. This matrix may function subsequently to trap proteins targeted to the membrane, resulting in the maintenance of membrane domains.

scholarly journals Novel Kelch-like Protein, KLEIP, Is Involved in Actin Assembly at Cell-Cell Contact Sites of Madin-Darby Canine Kidney Cells

2004 ◽  
Vol 15 (3) ◽  
pp. 1172-1184 ◽  
Author(s):  
Takahiko Hara ◽  
Hiroshi Ishida ◽  
Razi Raziuddin ◽  
Stephan Dorkhom ◽  
Keiju Kamijo ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cell Contact ◽  
Kidney Cells ◽  
Actin Assembly ◽  
Madin Darby Canine Kidney ◽  
Contact Sites ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
E Cadherin ◽  
Cell Cell

Dynamic rearrangements of cell-cell adhesion underlie a diverse range of physiological processes, but their precise molecular mechanisms are still obscure. Thus, identification of novel players that are involved in cell-cell adhesion would be important. We isolated a human kelch-related protein, Kelch-like ECT2 interacting protein (KLEIP), which contains the broad-complex, tramtrack, bric-a-brac (BTB)/poxvirus, zinc finger (POZ) motif and six-tandem kelch repeats. KLEIP interacted with F-actin and was concentrated at cell-cell contact sites of Madin-Darby canine kidney cells, where it colocalized with F-actin. Interestingly, this localization took place transiently during the induction of cell-cell contact and was not seen at mature junctions. KLEIP recruitment and actin assembly were induced around E-cadherin–coated beads placed on cell surfaces. The actin depolymerizing agent cytochalasin B inhibited this KLEIP recruitment around E-cadherin–coated beads. Moreover, constitutively active Rac1 enhanced the recruitment of KLEIP as well as F-actin to the adhesion sites. These observations strongly suggest that KLEIP is localized on actin filaments at the contact sites. We also found that N-terminal half of KLEIP, which lacks the actin-binding site and contains the sufficient sequence for the localization at the cell-cell contact sites, inhibited constitutively active Rac1-induced actin assembly at the contact sites. We propose that KLEIP is involved in Rac1-induced actin organization during cell-cell contact in Madin-Darby canine kidney cells.

scholarly journals Dissociation of Madin-Darby canine kidney epithelial cells by the monoclonal antibody anti-arc-1: mechanistic aspects and identification of the antigen as a component related to uvomorulin.

1985 ◽  
Vol 101 (4) ◽  
pp. 1307-1315 ◽  
Author(s):  
J Behrens ◽  
W Birchmeier ◽  
S L Goodman ◽  
B A Imhof
Keyword(s):  
Monoclonal Antibody ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Apical Cell ◽  
Junctional Complex ◽  
Madin Darby Canine Kidney ◽  
Surface Antibody ◽  
Darby Canine Kidney ◽  
Canine Kidney

It has previously been shown that the monoclonal antibody anti-Arc-1 dissociates Madin-Darby canine kidney (MDCK) epithelial cells and changes their morphology in vitro (Imhof, B.A., H.P. Vollmers, S.L. Goodman, and W. Birchmeier, 1983, Cell, 35:667-675). In this article we demonstrate that the anti-Arc-1 antibody recognizes an uvomorulin-like molecule on MDCK cells, i.e., it immunoprecipitates an 84-kD protein fragment from a tryptic digest of cell surfaces in the presence of Ca2+ (as does anti-uvomorulin antiserum). Furthermore, anti-uvomorulin antiserum prevents the binding of anti-Arc-1 to MDCK cells. The distribution of the Arc-1 antigen is also quite similar to that of uvomorulin: it is enriched at the cell-cell contacts both of MDCK cells and of cells in various canine tissues. In the intestinal epithelium the antigen could be further localized in the region of the junctional complex. To study the mechanism of action of the dissociating antibody, MDCK cells grown on Nuclepore filters in Boyden chambers were exposed to anti-Arc-1 from either the upper or lower compartment. It could be shown that the antibody interfered with cell adhesion only from the basolateral but not from the apical cell surface. Antibody action was inhibited in the presence of colchicine but not cytochalasin B. Furthermore, cell dissociation was prevented when the cellular cAMP level was raised. These findings indicate that the anti-Arc-1 antibody acts on a target below the tight junctions (possibly on the antigen located in the junctional complex), and they confirm that cytoskeleton and metabolic factors are actively involved in the maintenance of junctional integrity.

scholarly journals Depletion of E-Cadherin Disrupts Establishment but Not Maintenance of Cell Junctions in Madin-Darby Canine Kidney Epithelial Cells

2007 ◽  
Vol 18 (1) ◽  
pp. 189-200 ◽  
Author(s):  
Christopher T. Capaldo ◽  
Ian G. Macara
Keyword(s):  
Epithelial Cells ◽  
Mdck Cells ◽  
Morphological Changes ◽  
Cell Junctions ◽  
Cell Cortex ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
E Cadherin

E-cadherin forms calcium-dependent homophilic intercellular adhesions between epithelial cells. These contacts regulate multiple aspects of cell behavior, including the organization of intercellular tight junctions (TJs). To distinguish between the roles of E-cadherin in formation versus maintenance of junctions, Madin-Darby canine kidney (MDCK) cells were depleted of E-cadherin by RNA interference. Surprisingly, reducing E-cadherin expression had little effect on the protein levels or localization of adherens junction (AJ) or TJ markers. The cells underwent morphological changes, as the normally flat apical surface swelled into a dome. However, apical–basal polarity was not compromised, transmembrane resistance was normal, and zonula occludin protein 1 dynamics at the TJs were unchanged. Additionally, an E-cadherin/Cadherin-6 double knockdown also failed to disrupt established TJs, although β-catenin was lost from the cell cortex. Nevertheless, cells depleted of E-cadherin failed to properly reestablish cell polarity after junction disassembly. Recovery of cell–cell adhesion, transepithelial resistance, and the localization of TJ and AJ markers were all delayed. In contrast, depletion of α-catenin caused long-term disruption of junctions. These results indicate that E-cadherin and Cadherin-6 function as a scaffold for the construction of polarized structures, and they become largely dispensable in mature junctions, whereas α-catenin is essential for the maintenance of functional junctions.

Nonmitogenic morphoregulatory action of pp60v-src on multicellular epithelial structures

1987 ◽  
Vol 7 (4) ◽  
pp. 1326-1337
Author(s):  
S L Warren ◽  
W J Nelson
Keyword(s):  
Tyrosine Kinases ◽  
Mdck Cells ◽  
Growth Potential ◽  
Junctional Complex ◽  
Madin Darby Canine Kidney ◽  
Epithelial Monolayers ◽  
Zonula Occludens ◽  
Darby Canine Kidney ◽  
Canine Kidney

Madin-Darby canine kidney (MDCK) cells form polarized, multicellular epithelial structures in vitro. Low-level expression of pp60v-src in MDCK cells elicits plasticity in these multicellular structures. Plasticity was revealed by the displacement of cells from mechanically stressed regions of the epithelial monolayers; however, the two-dimensional relationship between the cells in the remainder of the monolayer was maintained. Electron microscopy of multicellular structures revealed abnormal separation of the lateral membranes of adjacent cells and selective uncoupling of the junctional complex; the zonula adherens was disrupted, but the zonula occludens and desmosomes were retained. Significantly, this result was not accompanied by transformation of the cells, as judged by the absence of anchorage-independent growth potential. These results demonstrate a nonmitogenic biological activity of pp60v-src which is experimentally dissociable from transformation. This morphoregulatory action on higher-order epithelial structures may reflect a function of related cellular tyrosine kinases.

Transepithelial Resistance and Inulin Permeability as Endpoints in In Vitro Nephrotoxicity Testing

2002 ◽  
Vol 30 (2_suppl) ◽  
pp. 53-59 ◽  
Author(s):  
Tracey Duff ◽  
Simon Carter ◽  
Gemma Feldman ◽  
Gordon McEwan ◽  
Walter Pfaller ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Electrical Resistance ◽  
Mdck Cells ◽  
Ussing Chamber ◽  
Fluorescein Isothiocyanate ◽  
Transepithelial Electrical Resistance ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

Transepithelial electrical resistance (RT) and the flux of fluorescein isothiocyanate (FITC) across Madin Darby canine kidney (MDCK) strain 1 cells and porcine epithelial kidney (LLC-PK1) monolayers were compared between three laboratories for a range of nephrotoxins. The precision of the REMS AutoSampler was similar to that of the Ussing chamber and the ENDOHM® technique, but superior to using chopstick electrodes, for measurements of resistance. The nephrotoxins used were selective for the proximal tubule, and in all cases, LLC-PK1 cells were more sensitive than MDCK cells. In most cases, change in RT was a more sensitive indicator of damage than alterations in FITC flux. The REMS system provides high intra-plate precision for RT measurements and is a higher throughput system, which is applicable to screening for nephrotoxicity in vitro.

scholarly journals Microtubule perturbation inhibits intracellular transport of an apical membrane glycoprotein in a substrate-dependent manner in polarized Madin-Darby canine kidney epithelial cells.

1990 ◽  
Vol 1 (12) ◽  
pp. 921-936 ◽  
Author(s):  
M J van Zeijl ◽  
K S Matlin
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Intracellular Transport ◽  
Mdck Cells ◽  
Sodium Dodecyl ◽  
Apical Plasma Membrane ◽  
Dependent Manner ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

The effects of microtubule perturbation on the transport of two different viral glycoproteins were examined in infected Madin-Darby canine kidney (MDCK) cells grown on both permeable and solid substrata. Quantitative biochemical analysis showed that the microtubule-depolymerizing drug nocodazole inhibited arrival of influenza hemagglutinin on the apical plasma membrane in MDCK cells grown on both substrata. In contrast, the microtubule-stabilizing drug taxol inhibited apical appearance of hemagglutinin only when MDCK cells were grown on permeable substrata. On the basis of hemagglutinin mobility on sodium dodecyl sulfate gels and its sensitivity to endo H, it was evident that nocodazole and taxol arrested hemagglutinin at different intracellular sites. Neither drug caused a significant increase in the amount of hemagglutinin detected on the basolateral plasma membrane domain. In addition, neither drug had any noticeable effect on the transport of the vesicular stomatitis virus (VSV)-G protein to the basolateral surface. These results shed light on previous conflicting reports using this model system and support the hypothesis that microtubules play a role in the delivery of membrane glycoproteins to the apical, but not the basolateral, domain of epithelial cells.

scholarly journals Myosin-1c regulates the dynamic stability of E-cadherin–based cell–cell contacts in polarized Madin–Darby canine kidney cells

2013 ◽  
Vol 24 (18) ◽  
pp. 2820-2833 ◽  
Author(s):  
Hiroshi Tokuo ◽  
Lynne M. Coluccio
Keyword(s):  
Dynamic Stability ◽  
Motor Function ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Cell Contacts ◽  
Cell Adhesions ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
E Cadherin ◽  
Cell Cell

Cooperation between cadherins and the actin cytoskeleton controls the formation and maintenance of cell–cell adhesions in epithelia. We find that the molecular motor protein myosin-1c (Myo1c) regulates the dynamic stability of E-cadherin–based cell–cell contacts. In Myo1c-depleted Madin–Darby canine kidney cells, E-cadherin localization was dis­organized and lateral membranes appeared less vertical with convoluted edges versus control cells. In polarized monolayers, Myo1c-knockdown (KD) cells were more sensitive to reduced calcium concentration. Myo1c separated in the same plasma membrane fractions as E-cadherin, and Myo1c KD caused a significant reduction in the amount of E-cadherin recovered in one peak fraction. Expression of green fluorescent protein (GFP)–Myo1c mutants revealed that the phosphatidylinositol-4,5-bisphosphate–binding site is necessary for its localization to cell–cell adhesions, and fluorescence recovery after photobleaching assays with GFP-Myo1c mutants revealed that motor function was important for Myo1c dynamics at these sites. At 18°C, which inhibits vesicle recycling, Myo1c-KD cells accumulated more E-cadherin–positive vesicles in their cytoplasm, suggesting that Myo1c affects E-cadherin endocytosis. Studies with photoactivatable GFP–E-cadherin showed that Myo1c KD reduced the stability of E-cadherin at cell–cell adhesions. We conclude that Myo1c stabilizes E-cadherin at adherens junctions in polarized epithelial cells and that the motor function and ability of Myo1c to bind membrane are critical.

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